Increased pulmonary vascular resistance in pulmonary hypertension (PH) is caused by vasoconstriction and obstruction of small pulmonary arteries by proliferating vascular cells. In analogy to cancer, subsets of proliferating cells may be derived from endothelial cells transitioning into a mesenchymal phenotype. To understand phenotypic shifts transpiring within endothelial cells in PH, we injected rats with alkaloid monocrotaline to induce PH and measured lung tissue levels of endothelial-specific protein and critical differentiation marker vascular endothelial (VE)-cadherin. VE-cadherin expression by immonoblotting declined significantly 24 h and 15 days postinjection to rebound to baseline at 30 days. There was a concomitant increase in transcriptional repressors Snail and Slug, along with a reduction in VE-cadherin mRNA. Mesenchymal markers α-smooth muscle actin and vimentin were upregulated by immunohistochemistry and immunoblotting, and α-smooth muscle actin was colocalized with endothelial marker platelet endothelial cell adhesion molecule-1 by confocal microscopy. Apoptosis was limited in this model, especially in the 24-h time point. In addition, monocrotaline resulted in activation of protein kinase B/Akt, endothelial nitric oxide synthase (eNOS), nuclear factor (NF)-κB, and increased lung tissue nitrotyrosine staining. To understand the etiological relationship between nitrosative stress and VE-cadherin suppression, we incubated cultured rat lung endothelial cells with endothelin-1, a vasoconstrictor and pro-proliferative agent in pulmonary arterial hypertension. This resulted in activation of eNOS, NF-κB, and Akt, in addition to induction of Snail, downregulation of VE-cadherin, and synthesis of vimentin. These effects were blocked by eNOS inhibitor N(ω)-nitro-l-arginine methyl ester. We propose that transcriptional repression of VE-cadherin by nitrosative stress is involved in endothelial-mesenchymal transdifferentiation in experimental PH.

Embryonic Stem (ES) or induced Pluripotent Stem (iPS) cells are important sources for cardiomyocyte generation, targeted for regenerative therapies. Several in vitro protocols are currently utilized for their differentiation, but the value of cell-based approaches remains unclear. Here, we characterized a cardiovascular progenitor population derived during ES differentiation, after selection based on VE-cadherin promoter (Pvec) activity. ESCs were genetically modified with an episomal vector, allowing the expression of puromycin resistance gene, under Pvec activity. Puromycin-surviving cells displayed cardiac and endothelial progenitor cells characteristics. Expansion and self-renewal of this cardiac and endothelial dual-progenitor population (CEDP) were achieved by Wnt/β-catenin pathway activation. CEDPs express early cardiac developmental stage-specific markers but not markers of differentiated cardiomyocytes. Similarly, CEDPs express endothelial markers. However, CEDPs can undergo differentiation predominantly to cTnT+ (~47%) and VE-cadherin+ (~28%) cells. Transplantation of CEDPs in the left heart ventricle of adult rats showed that CEDPs-derived cells survive and differentiate in vivo for at least 14 days after transplantation. A novel, dual-progenitor population was isolated during ESCs differentiation, based on Pvec activity. This lineage can self-renew, permitting its maintenance as a source of cardiovascular progenitor cells and constitutes a useful source for regenerative approaches.

Vascular Endothelial cadherin, a type II classical cadherin, is the major cadherin molecule participating in homotypic cell-cell adhesion structures between endothelial cells. It associates with cytoplasmic and membrane cytoskeletal elements to form endothelial adherens junctions (AJs), pivotal in regulating endothelial barrier function in the adult. VE-cadherin-mediated AJs are also involved in signaling via direct or indirect associations with receptors. The generation of mutant animals, especially mice and zebrafish, revealed many details about the role of VE-cadherin-mediated AJs in cardiovascular development. In general, VE-cadherin knockout (KO) in mice is embryonic lethal due to severe cardiovascular defects and major signaling pathways as well as vascular formation cues were discovered in developing endothelium. However, there is little information regarding AJs formation and their components in cardiovascular progenitors. We have characterized in detail the activation pattern of mouse VE-cadherin promoter (Pvec) in a mouse embryonic stem cells (ESCs) differentiation system in vitro. Surprisingly, we found that it is activated transiently in cardiac progenitors that belong to the second heart field. Based on Pvec activation we isolated this population in vitro and found that it can self-renew by induction of the Wnt/β-catenin pathway. Next, we successfully established cell culture conditions that allowed self-renewal of this population that consists of endothelial and cardiac progenitors. Transplantation in rat hearts showed that they can survive and differentiate to cardiomyocytes and endothelial cells. Although further characterization is needed, these cells can be used in cell-based therapies as well as in drug screening.

Peptide analogs/conjugates Abbreviation Ac-(Lys-Aib-Cys) 4 -NH 2 CPSOC CF-Ahx-[Lys-Aib-Cys(S-CH 2 CONH 2 )] 4 -NH 2 CF-Ahx-CPSOC(S-CH 2 CONH 2 ) CF-G 48 RKKRRQRRRPPQ 60 -Cys (CH 2 CONH 2 )-NH 2 CF-Tat 48-60 -Cys (CH 2 CONH 2 ) CF-Ahx-S 158 ALTQKGLKNVFDEA 172 -NH 2 CF-Ahx-Cdc42 158-172 CF-Ahx-[Lys-Aib-Cys(S-CH 2 CO-V 181 IKKSKK 187 -NH 2 )] 4 -NH 2 CF-Ahx-CPSOC(S-CH 2 CO-Cdc42 181-187 ) CF-Ahx-[Lys-Aib-Cys(S-CH 2 CO-I 173 VAALEPPVIKKSKK 187 -NH 2 )] 4 -NH 2 CF-Ahx-CPSOC(S-CH 2 CO-Cdc42 173-187